JP3491036B2 - Cyclic nucleotide phosphodiesterase inhibitor - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a water-soluble lignin extracted from a mushroom or a medium after cultivating the mushroom,
The effect of water-soluble lignin extracted from cereal bran on cyclic AMP phosphodiesterase.

[0002]

2. Description of the Related Art Lignin is one of the most abundant organic substances in nature as a main component of wood, along with cellulose and hemicellulose. However, lignin sulfonic acid produced by using a cooking eluate produced as a by-product during pulp production is an unused natural resource that is mainly used as an industrial dispersant. In addition, lignin can be extracted from ascomycetes and basidiomycetes as well as cereal bran, and effective utilization of abundant natural organic substances has been a major issue. In addition, beech shimeji and the like are artificially cultivated in a medium containing sawdust and the like as a main component, but effective use of the medium after harvest has been a problem. On the other hand, it is known that the lignin fraction of extracts such as pine cones and mycelia of various mushrooms has anti-HIV-1 activity. Other lignin-like substances have been reported to have physiological activities such as promotion of cytokine production by peripheral blood mononuclear cells, activation of macrophages, and suppression of plaque formation by influenza virus and herpes simplex virus in cultured cell lines. Furthermore,
It has been confirmed that synthetic lignin, which is a polymer produced by dehydrogenative polymerization of p-coumaric acid, ferulic acid, and caffeic acid, which are the constituents of lignin, in the presence of H2O2 with horseradish peroxidase also has anti-HIV-1 activity. (Japanese clinical, Vol.51,
p.127 (1993)). However, these have not yet been put to practical use, and cyclic nucleotide phosphodiesterase inhibition has not been reported. Cyclic AMP (cyclic adenosine 3 ', 5'-monophosphate, cyclic AMP, cAM
P) is a second messenger that transmits information on many hormones and neurotransmitters to intracellular target molecules, and is synthesized from ATP by adenylate cyclase existing in the cell membrane. Cyclic AMP is widely present in the bacteria and animal kingdoms. In higher animals, various hormones activate receptors, G proteins, and adenylate cyclase, and cyclic
Promotes the production of AMP. The point of action of cyclic AMP in animals is the cyclic AMP-dependent protein kinase (A
A-kinase uses many intracellular proteins as substrates, and cyclic AMP regulates various cell functions. For example, it is known that A kinase phosphorylates the transcription factor CREB (cyclic AMP response element binding protein), and phosphorylated CREB binds to CRE (cyclic AMP response element) and regulates gene transcription activity. ing.

Cyclic AMP having such a function
Is degraded to 5'-AMP by cyclic nucleotide phosphodiesterase. Cyclic nucleotide phosphodiesterase, along with adenylate cyclase,
It is thought to regulate the quantitative level of intracellular cyclic AMP. Cyclic nucleotide phosphodiesterase is present in various tissues such as brain, lung, liver and heart, and binds to calmodulin (activator),
There are several types of enzymes with different affinities for substrates. Thus, it is considered possible to control various biological functions by controlling the concentrations of cyclic AMP by controlling the activities of adenylate cyclase, cyclic nucleotide phosphodiesterase and the like. For example, in the inflammatory process of the lungs, humoral factors act on vascular endothelial cells, and proteinaceous fluid in blood easily enters the interstitium through the intercellular spaces, resulting in edema. Increased cyclic AMP concentration in vascular endothelial cells has been reported to suppress such endothelial permeability (Am. J. Physio
L., Vol.275, p.L203 (1998)). Many purine alkaloid compounds such as theophylline have been reported as substances that inhibit cyclic nucleotide phosphodiesterase, but such activity has not been reported for lignin-related substances.

[0004]

[Means for Solving the Problems] The present inventors have aimed to effectively and highly utilize lignin, which is abundant in nature, as a drug or a food material for specified health use.
As a result of evaluating various physiological activities using animal cells, it was found that water-soluble lignin and the like have a cyclic nucleotide phosphodiesterase inhibitory action, and the present invention was completed. That is, the present invention is (a) a water-soluble lignin having a cyclic nucleotide phosphodiesterase inhibitory action. Hereinafter, the present invention will be described in detail.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION The water-soluble lignin-like substance according to the present invention is contained in particularly large amounts in xylem of vascular plants, ascomycetes, and basidiomycetes, and p-coumaryl alcohol, coniferyl alcohol, cinnamon It is an amorphous polyphenol in which phenylpropenoid such as pill alcohol is used as a structural unit and is polycondensed in a complicated manner. Although natural lignin is water-insoluble, it becomes water-soluble by alkali treatment, etc., and ascomycetes and basidiomycetes lower the molecular weight of woody or herbaceous lignin due to the action of the enzyme that they have, making it a water-soluble lignin-like substance. Can be converted. Extraction by alkali treatment can be carried out according to the method described in, for example, Cereal Chem. Vol.66, p.289 (1989). In addition, ascomycetes and lignin-like substances converted by basidiomycetes are, for example,
It can be recovered by extraction with 100 ° C hot water.
Furthermore, water-soluble lignin-like substances were ferulic acid, p-coumaric acid, horseradish peroxidase in the presence of H2O2.
It is also possible to synthesize by dehydrogenative polymerization of caffeic acid. Dehydrogenative polymerization with peroxidase is described, for example, in Che
m. Pharm. Bull. Vol.39, p.950 (1991). Below, the cyclic of the present invention
A method for producing an AMP phosphodiesterase inhibitory substance from a medium after cultivating Buna shimeji will be specifically described, but such a substance should be similarly produced from other ascomycetes or basidiomycetes themselves, or a medium after culturing them. You can Such fungi include shiitake mushrooms and matsutake mushrooms. In addition, as a medium for cultivating such fungi, the mixing ratio of sawdust, rice bran, general bran, and okara as the main components is not limited. Water is added to the medium after the cultivation of Bunashimeji and the mixture is heat-treated at 80 to 100 ° C for 5 to 60 minutes, and then centrifuged to collect the supernatant. To this, an inorganic acid such as dilute hydrochloric acid is added to adjust the pH to 1-2, the resulting precipitate is suspended in water, and an alkaline solution is added to make it neutral. This solution is filtered through a low-molecular ultrafiltration membrane (for example, YM-1 (Amicon)), the residue is thoroughly washed with water, and then dried under reduced pressure.

In order to extract the lignin-like substance according to the present invention from wheat bran, the bran is first subjected to a degreasing treatment such as washing with acetone. Next, defatted bran was added to a phosphate buffer containing α-amylase,
By treating for 30 minutes or more at ~ 100 ℃, preferably 70
It is subjected to sugar decomposition treatment by treating at 90 ° C for 1 hour or more. Next, saccharide-degrading bran is added to sodium acetate buffer containing protease and treated at 30 to 90 ° C. for 1 hour or longer, preferably at 40 to 60 ° C. for 5 hours or longer to be proteolytically treated. It The thus treated enzyme-treated bran is subjected to alkali extraction treatment by a standard method. For example, it is carried out by adding enzyme-treated bran to a sodium hydroxide solution and conducting an extraction treatment at 20 to 50 ° C for 30 minutes or longer, preferably at 30 to 40 ° C for 1 hour or longer. The extracted lignin is acid-precipitated and collected by centrifugation. The cyclic nucleotide phosphodiesterase inhibitor of the present invention may be formulated as it is or in the form of tablets, powders, wettable powders, emulsions, capsules, etc., which are commonly used solid carriers, liquid carriers, emulsifying dispersants and the like. It can be used as a cyclic nucleotide phosphodiesterase inhibitor. As the carrier,
Water, gelatin, starch, magnesium stearate, lactose, vegetable oil and the like can be mentioned. In addition, the substance of the present invention suppresses endothelial hyperpermeability, or cyclic AMP
In addition to being used as a medicine for substituting hormones, which is used as a second messenger, it can be added to various foods and used as functional foods, foods for specified health uses, and the like. Such foods include soft drinks, lactic acid drinks, soups,
Examples include cheese, ham, and confectionery. The substance of the present invention can also be added to pet foods, feeds, etc. to substitute hormones for pets and livestock to improve health. Examples of the usage form of the cyclic nucleotide phosphodiesterase inhibitor containing the lignin-like substance of the present invention as an active ingredient include parenteral administration such as intravenous injection and rectal administration, or oral administration. The content of the lignin-like substance of the present invention in the cyclic nucleotide phosphodiesterase inhibitor can be appropriately changed depending on its administration route and dosage form,
For example, the substance of the present invention is contained in an amount of 5 to 90% by weight, preferably 10 to 60% by weight, based on the total weight of the preparation. When added to a functional food or a food for specified health use, for example, the lignin-like substance of the present invention in the food is 0.01 to 50.
It is contained by weight%.

[0007]

EXAMPLES Examples of the cyclic AMP phosphodiesterase inhibitor according to the present invention will be described below. However, it is needless to say that the present invention is not limited to such embodiments, and there are various embodiments within the scope of the technical matters of the claims. Example 1 (Extraction of lignin from the medium after cultivating beech shimeji) After harvesting beech shimeji cultivated in a medium for cultivating beech shimeji containing sawdust, rice bran, general bran, and okara as a main component, water was added to 1.5 kg of the medium. Add 1.3 liters, 15
It was kept at 100 ° C for a minute. Centrifugation was repeated twice and the supernatant was collected. This solution was adjusted to pH 1 with diluted hydrochloric acid, the resulting precipitate was suspended in a small amount of water and diluted with diluted sodium hydroxide.
The pH was adjusted to around 7. As a result, the precipitate was completely dissolved. This solution was filtered through an ultrafiltration membrane YM-1 (Amicon), and the residue was thoroughly washed with water and dried under reduced pressure. This gave 0.5 g of lignin-like substance.

Example 2 (Extraction of lignin from wheat bran) PBSchwarz et al. (Cereal Chem. Vol.66, p.289 (1989))
Alkaline lignin was extracted and purified from wheat bran according to the method of 1. That is, wheat bran 30g and acetone 250ml
And degreased. This operation was repeated twice more, and the bran was dried under reduced pressure. This defatted wheat bran was placed in a flask, phosphate buffer (50 mM, pH 7.5) 700 ml, α-amylase (from Bacillus licheniformis, Sigma)
300 mg was added and the temperature was raised to 90 ° C. with stirring. Then add 150 ml of phosphate buffer (50 mM, pH 7.5),
The mixture was stirred at 85 ° C for 1 hour. After that, it was transferred to a water bath at 45 ° C, and a protease (from Streptomyces griseus, manufactured by Sigma)
62 mg was added and stirred at 45 ° C. for 18 hours. After reaction, 8,000r
The precipitate was collected by centrifugation at pm. The precipitate was again dispersed in water and centrifuged at 8,000 rpm to collect the precipitate. This washing operation was repeated twice more. To the washed precipitate, 900 ml of sodium acetate buffer (50 mM, pH 4.5) was added, 120 mg of amyloglucosidase (derived from Rhizopus, manufactured by Sigma) was further added, and the mixture was stirred at 45 ° C for 20 hours. After the reaction, the product was washed in the same manner as described above to obtain a precipitate, which was dried under reduced pressure.
450 ml of 1N sodium hydroxide was added to the whole amount of wheat bran thus enzyme-treated, and the mixture was heated at 30 ° C. under a nitrogen gas stream for 2
Stir for hours. Then, the insoluble matter was removed by centrifugation at 8,000 rpm. Concentrated hydrochloric acid was gradually added to the supernatant solution to precipitate lignin, which was then centrifuged to recover lignin. 70 ml of dioxane was added to this lignin, and the mixture was stirred for 2 days to dissolve the lignin in dioxane. The insoluble matter was removed by centrifugation at 12,000 rpm, and the supernatant was concentrated to 10 ml by a rotary evaporator.
This solution was added dropwise to 120 ml of 1M acetic acid to obtain a precipitate. The precipitate was dried under reduced pressure to remove acetic acid and water. The purified lignin obtained was approximately 100 mg of brown powder. It was confirmed from the result of measurement by Fourier transform infrared spectroscopy that this powder had a lignin structure.

Example 3 (Cyclic nucleotide phosphodiesterase inhibitory action) The cyclic nucleotide phosphodiesterase inhibitory action was measured by the following method. Bovine brain-derived phosphodiesterase activator (calmodulin) purchased from Sigma was dissolved in a buffer solution (62.5 mM Tris-HCl buffer, pH 7.5, 6.3 mM magnesium chloride, 3.8 mM β-mercaptoethanol, 0.038 mM calcium chloride), and 1
The activator solution was 4.3 units / ml. In addition, bovine brain-derived phosphodiesterase purchased from Sigma was dissolved in the above buffer solution at a concentration of 0.1 unit / ml to prepare an enzyme solution. A 1 mM cyclic AMP aqueous solution was used as a substrate solution.
In a 0.5 ml capacity plastic tube, put activator solution 140 μl, enzyme solution 20 μl, measurement sample solution 20 μl,
After incubating at 30 ° C. for 5 minutes, 20 μl of the substrate solution was added and mixed well, and the reaction was carried out at 30 ° C. for 40 minutes. The reaction was then stopped by adding 20 μl of 10% trifluoroacetic acid. After the reaction was stopped, the produced AMP was quantified by high performance liquid chromatography under the following conditions. High Performance Liquid Chromatography Measurement Conditions Column: μBondasphere 5 μC ₁₈ 300 Å (Waters
3.9 x 150 mm) Eluent: linear gradient of 0-63% acetonitrile containing 0.1% trifluoroacetic acid (20 minutes) Flow rate: 1 ml / min Detection: UV absorption at 260 nm Repeated these experiments several times, The inhibition rate was calculated by the following formula. Inhibition rate = (A-B) / AX 100 (%) [wherein, A: peak area of AMP without inhibitor, B: peak area of AMP when inhibitor is added]
The peptide concentration at which the above inhibition rate was 50% was expressed as an IC ₅₀ value. The IC ₅₀ values thus obtained are shown in Table 1.

[0010]

[Table 1]

[0011]

The cyclic nucleotide phosphodiesterase inhibitor of the present invention has the following effects. (1) It has a cyclic nucleotide phosphodiesterase inhibitory action. (2) In addition to humans, substitutes for hormones that use cyclic AMP as a second messenger in pets such as dogs and cats, and industrial animals such as cows, pigs, and horses, and enhances endothelial permeability due to inflammation of pulmonary vascular endothelium It can be used for the purpose of improving health, such as suppressing. (3) The cyclic nucleotide phosphodiesterase inhibitor of the present invention can be produced at a lower cost than vascular plants, ascomycetes and basidiomycetes which are abundant in nature.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI A61P 29/00 A61P 29/00 43/00 111 43/00 111 C12N 9/99 C12N 9/99 // C12N 1/14 1 / 14 G (56) Reference JP-A-52-136193 (JP, A) Chem. Pharm. Bull. , 1981, Vol. 29, No. 12, pp. 3586-3592 (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K 35/78 A61K 35/70 A61K 35/84

Claims (3)

(57) [Claims] 1. Water and alkali are added to enzyme-treated wheat bran and the mixture is stirred under an inert gas stream, insoluble matter is removed, and an inorganic acid is added to the supernatant to precipitate the mixture, which has a molecular weight of 1000.
A cyclic nucleotide phosphodiesterase inhibitor comprising a lignin-like substance obtained by ultrafiltration of the following and further washing the solubilized lignin-like substance after centrifugation. 2. The enzyme is α-amylase, protease,
The cyclic nucleotide phosphodiesterase inhibitor comprising a water-solubilized lignin-like substance according to claim 1, which is any one kind or two or more kinds of amyloglucosidase. 3. Ascomycetes and basidiomycetes are made to have a low molecular weight of woody or herbaceous lignin by the action of the enzymes possessed by them, and those having a molecular weight of 1000 or less are ultrafiltered to further wash the water-solubilized lignin-like substance. Ligni obtained by
Cyclic nucleotide phosphodiesterase inhibitor consisting of a phosphonate- like substance .